One night of disturbed sleep is all that it takes for us to realize how important normal sleep / wake rhythms are for mental and physical health. These rhythms are controlled by an internal circadian (~24hr) clock, and we have learnt most about how the clock functions from studies in Drosophila. Indeed, the first human sleep disorder gene identified is a homologue of the period clock gene, first identified and cloned in Drosophila. As a simple behavior, circadian rhythms provide the opportunity to understand fuindamental mechanisms of how brains control behavior. In particular, the Drosophila clock is an excellent system to understand how transcription factors dynamically regulate neuronal function, a topic of broad general importance in Neuroscience. Here, we propose to study how clock transcription factors control clock neuron activity. We focus on Vrille (VRI) and PDP1, which comprise the second Drosophila clock feedback loop. VRI and PDP1 have roles in maintaining the robustness of the feedback loops themselves, and are the most downstream factors that link the clock to rhythmic outputs by regulating genes that peak at dawn. In Aim 1, we propose a detailed analysis of the VRI/PDP1 clock loop. We have identified that additional regulators exist in this loop. One regulator is Cryptochrome (CRY), previously characterized as a circadian photoreceptor, but recently established by my lab to also function as a transcriptional represser in most clock neurons. Here we propose to study cry regulation by PDP1 in different clock neurons. A second regulator of vri and Pdp1 expression may be Stich1, a homologue of the mammalian Dec clock proteins, which are potent transcriptional repressors, but have not been formally fitted into the clockworks. Finally the identification of a cry null allele will allow us to test in which clock neurons in the brain CRY is a repressor. In Aim 2, we seek to extend exciting findings that should help link the clock to output pathways. We have developed a behavioral genomics approach in which gene expression from purified pacemaker neurons can be assayed across the whole genome. We have currently analyzed different times of day, but we will extend this analysis to different clock mutants with known effects on the outputs of pacemaker neurons. Thus we can correlate patterns of gene expression with known behavioral outputs. In our initial studies, we identified a group of genes which may underlie rhythmic neuronal activity and neurotransmission from pacemaker neurons. We propose to study mutants in these genes for defects in circadian behaviors. Many previously unstudied genes are also tightly clock-regulated, and we will narrow done which to study further through additional GeneChip experiments in vri and Pdp1 mutants.